Replication-competent infectious hepatitis B virus vectors carrying substantially sized transgenes by redesigned viral polymerase translation.
Replication-competent infectious hepatitis B virus vectors carrying substantially sized transgenes by redesigned viral polymerase translation.
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通过重新设计的病毒聚合酶翻译，具有复制能力的传染性乙型肝炎病毒载体携带大量转基因。
DOI：
10.1371/journal.pone.0060306
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发表时间：
2013
期刊：
影响因子：
3.7
通讯作者：
Sun D
中科院分区：
文献类型：
--
作者：
Wang Z;Wu L;Cheng X;Liu S;Li B;Li H;Kang F;Wang J;Xia H;Ping C;Nassal M;Sun D
病毒载体是能够将非病毒遗传信息传递到细胞中的工程病毒变体，通常通过与亲本病毒相同的途径。对于几个病毒家族，携带报告基因的可复制载体已经成为用于简单和定量监测复制和感染的宝贵工具，并且因此也用于鉴定抗病毒药物和病毒易感细胞。对于B型肝炎病毒（HBV），一种引起B型肝炎的小包膜DNA病毒，这样的载体是不可用的，因为插入到其微小的3.2kb基因组中几乎不可避免地影响必需的复制元件。HBV通过前基因组（pg）RNA的逆转录进行复制，前基因组RNA也是衣壳（核心）蛋白和逆转录酶（Pol）的双顺反子mRNA所需的;它们的开放阅读框（ORF）重叠约150个碱基对。下游Pol ORF的翻译不涉及常规的内部核糖体进入位点（IRES）。我们推断，复制重叠区域并为Pol和中间插入的转基因的翻译提供人工IRES控制可能产生功能性三顺反子pgRNA，而不干扰包膜蛋白表达。作为IRES，我们使用称为Rbm 3 IRES的22个核苷酸元件来最小化基因组大小的增加。模型质粒证实其活性，即使在三顺反子安排。携带399 bp和720 bp杀稻瘟菌素抗性转基因（BsdR）和人源化海肾绿色荧光蛋白（hrGFP）的完整HBV基因组的类似质粒产生与野生型HBV相似的核心和包膜蛋白;而hrGFP载体复制较差，BsdR载体产生的复制DNA约为野生型HBV的40%。然而，这两种载体都形成了对HBV敏感的HepaRG细胞具有感染性的包膜病毒体。由于有许多大小约为500 bp或更小的报告基因和效应基因，新的HBV载体应该成为更好地了解和对抗这种重要病原体的非常有用的工具。
Viral vectors are engineered virus variants able to deliver nonviral genetic information into cells, usually by the same routes as the parental viruses. For several virus families, replication-competent vectors carrying reporter genes have become invaluable tools for easy and quantitative monitoring of replication and infection, and thus also for identifying antivirals and virus susceptible cells. For hepatitis B virus (HBV), a small enveloped DNA virus causing B-type hepatitis, such vectors are not available because insertions into its tiny 3.2 kb genome almost inevitably affect essential replication elements. HBV replicates by reverse transcription of the pregenomic (pg) RNA which is also required as bicistronic mRNA for the capsid (core) protein and the reverse transcriptase (Pol); their open reading frames (ORFs) overlap by some 150 basepairs. Translation of the downstream Pol ORF does not involve a conventional internal ribosome entry site (IRES). We reasoned that duplicating the overlap region and providing artificial IRES control for translation of both Pol and an in-between inserted transgene might yield a functional tricistronic pgRNA, without interfering with envelope protein expression. As IRESs we used a 22 nucleotide element termed Rbm3 IRES to minimize genome size increase. Model plasmids confirmed its activity even in tricistronic arrangements. Analogous plasmids for complete HBV genomes carrying 399 bp and 720 bp transgenes for blasticidin resistance (BsdR) and humanized Renilla green fluorescent protein (hrGFP) produced core and envelope proteins like wild-type HBV; while the hrGFP vector replicated poorly, the BsdR vector generated around 40% as much replicative DNA as wild-type HBV. Both vectors, however, formed enveloped virions which were infectious for HBV-susceptible HepaRG cells. Because numerous reporter and effector genes with sizes of around 500 bp or less are available, the new HBV vectors should become highly useful tools to better understand, and combat, this important pathogen.
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影响因子：
3.7
作者：
Edmonds TG;Ding H;Yuan X;Wei Q;Smith KS;Conway JA;Wieczorek L;Brown B;Polonis V;West JT;Montefiori DC;Kappes JC;Ochsenbauer C
通讯作者：
Ochsenbauer C
影响因子：
3.5
作者：
Haase R;Argyros O;Wong SP;Harbottle RP;Lipps HJ;Ogris M;Magnusson T;Vizoso Pinto MG;Haas J;Baiker A
通讯作者：
Baiker A
DOI：
10.1073/pnas.0710650105
发表时间：
2008-03-25
影响因子：
11.1
作者：
Baranick, Brian T.;Lemp, Nathan A.;Logg, Christopher R.
通讯作者：
Logg, Christopher R.
影响因子：
3.7
作者：
Dhason MS;Wang JC;Hagan MF;Zlotnick A
通讯作者：
Zlotnick A
影响因子：
5.4
作者：
BETT, AJ;PREVEC, L;GRAHAM, FL
通讯作者：
GRAHAM, FL